The steroidogenic acute regulatory protein (StAR) mediates trophic hormone-stimulated steroid biosynthesis. Since steroid hormones play critical roles in maintaining body homeostasis, the mechanism regulating StAR gene expression is an important component of research on trophic hormone-induced steroidogenesis. It is well known that trophic hormone stimulation induces cyclic AMP (cAMP) formation followed by activation of protein kinase A (PKA) which, through as yet unknown mechanisms, regulate StAR gene expression. However, trophic hormones also induce arachidonic acid release, and inhibition of arachidonic acid release inhibited StAR gene expression and steroidogenesis in spite of high intracellular levels of cAMP. While the mechanism of arachidonic acid regulation of StAR gene expression is not clear, recent studies suggested a scenario in which arachidonic acid and cAMP transduce signals through two separate pathways and co-regulate the transcription factors acting on the StAR promoter. To study this possible mechanism and to prove the co-regulation of these two pathways in StAR gene expression, the following specific Aims are proposed: 1) to demonstrate that in trophic hormone-stimulation, both the arachidonic acid and cAMP-PKA- phosphorylationpathways are required with neither one along being sufficient for StAR gene expression and to study roles of each pathway in steroidogenesis; 2) to determine which arachidonic acid metabolites are involved in trophic hormone-stimulated StAR gene expression and to examine the roles of these metabolites in steroidogenesis; 3) to construct StAR promoter/luciferase reporter plasmids containing serial deletions of the wild type StAR promoter and to test promoter activities with the goal of selecting the region responding to arachidonic acid and identifying the arachidonic acid-response element (ARE) in the selected promoter sequences by continueous deletion and mutagenesis. The resulting ARE DNA sequences will be used in future studies to identify arachidonic acid-regulated transcription factors which may participate in StAR gene transcription. The long term goal of the project is to understand the molecular mechanism responsible for arachidonic acid regulation of hormone-stimulated steroid biosynthesis and the role of this fatty acid in steroid hormone-related physiological activities and diseases including gonadal steroid-related diseases in reproduction.